Anti rabbit igg

Total protein from left ventricle tissue samples was extracted, as described above for the preparation of serum and tissue samples, and measured using a BCA protein assay kit. Following heating at 95°C for 5 min, 20 µg/sample lysates were separated by 10 or 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% fat-free milk in TBST buffer (0.1% Tween-20 in TBS) for 90 min at room temperature, and subsequently incubated with primary antibodies against profilin-1 (cat. no. ab124904, 1:1,500 dilution), p65 (cat. no. BS1253, 1:1,000 dilution), RAGE (cat. no. sc-365154, 1:600 dilution), Rho (cat. no. ab40673, 1:1,000 dilution) and GAPDH (cat. no. G9545, 1:5,000 dilution) at 4°C overnight. Following primary antibody incubation, membranes were subsequently washed and incubated with horseradish peroxidase-conjugated goat anti-mouse (cat. no. CW0102, 1:5,000 dilution; CW Biotech, Co., Ltd., Beijing, China) or anti-rabbit IgG (cat. no. CW0103, 1:5,000 dilution; CW Biotech) secondary antibodies for 1 h at room temperature. Potent ECL kit (cat. no. 70-P1425; MultiSciences Biotech Co., Ltd., Hangzhou, China) was used to visualize proteins. The signal was detected and ratios of the target protein against the GAPDH control were calculated using the Image Lab software (version 5.2.1; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

To obtain cell and tissue proteins, samples were processed with 2% sodium dodecyl sulfate (SDS) lysis buffer and sonicated to break up DNA. Lysates were boiled for 10 min at 98°C. Then, the samples were measured by BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China), and 20 μg of total protein was loaded. Transferred polyvinylidene fluoride membranes were incubated with primary antibodies against H3K4me3 (1:1000; #GC-263, PTMbiolabs, Chicago, IL, USA), MLL1 (1:1000; #14197, Cell Signaling Technology, Danvers, MA, USA), and β-tubulin (1:3000; Beyotime Institute of Biotechnology) overnight at 4°C, followed by incubation with secondary antibodies of anti-mouse IgG and anti-rabbit IgG, respectively (1:2000; CWbiotech, Beijing, China) for 1 h at room temperature. Western blot analyses were normalized to β-tubulin. The blots were developed with Super Signal Pico substrate (Pierce Biotechnology, Shanghai, China). Each immunoblot was repeated three times, with samples obtained from different experiments. The relative intensity of protein bands was measured with NIH image J software.